Genotyping For Three Short Tandem Repeat Loci Using Next Generation Sequencing Method

BackgroundCurrently,polymerase chain reaction-capillary electrophoresis(PCR-CE)technology is still the mainstream assay to apply for genotyping of short tandem repeat(STR)genetic markers in forensic science.STR genotyping based on PCR-CE only is achieved by the length polymorphism,so the alleles of STR with same length but different motif are usually analyzed to be as the same genotype.Moreover,PCR-CE based for allele typing for STR loci easily generate multiple interference peaks such as off-ladder peaks due to electrophoretic drift and other factors that could further trigger the wrong genotyping for STR markers.However,Next generation sequence(NGS)as a new sequence technique not only analyze the length polymorphism but also differentiate its sequence polymorphism of STR loci which have the same length,which makes a STR locus get more genetic polymorphism information.Additionally,some interference peaks could be avoided due to the direct sequence for alleles by NGS method.ObjectiveHenan Han population was recruited as the study subject.One autosomal STR locus D8S1179,two multi-copy Y-STR loci including DYF404S1a/b(2 copies)and DYF399S1a/b/c(3 copies)which belong to rapidly mutating(RM)Y-STR were selected for genotyping by using NGS technique.Firstly,genotyping of D8S1179 locus were optimized for establishment of protocol of NGS technique.Comparing NGS to PCR-CE technique was aimed to genotype for two multi-copy Y-STR loci and to obtain more genetic polymorphism information,also to provide the technical support and basic data for applications in forensic medicine and study of human genetics.Methods1.Sample collection and whole genomic DNA extraction:In the case of informed consent,225 blood samples of unrelated male individuals were collected from Henan Han population;9947A(female)and 9948(male)were used as positive control DNA.Ultrapure distilled water was used as a negative control.Genomic DNA was extracted by saturated phenol-chloroform method.2.Fluorescence amplification and genotyping based PCR-CE technique:DYF404S1 and DYF399S1 loci were labeled with 6-FAM(blue)fluorescent dye,and their PCR products were seperated on ABI3130XL genetic analyzer,and genotyping was performed by GeneMapper ID v3.2 software.3.Nomenclature for alleles and preparation of making allelic ladders:Different alleles of the three STR loci was sequenced.The nomenclature for alleles was based on the recommendations of the Internation Society of Forensic Genetics(ISFG)Commossion.Allelic ladders were performed by gels recycling protocol followed by 6%polyacrylamide gel electrophoresis.4.Primer design and library construction for NGS:Three pairs of STR-locus-specific primers were designed by using Primer 5 software.Adapters were added at both ends of the primers.After the synthesis of the NGS primers,primers for NGS were verified by general PCR followed with agarose gel electrophoresis.25 pairs of NGS primers of DYF404S1 and DYF399S1 loci and 132 pairs for locus D8S1179 were obtained finally.The BIOO RAPID DNA Genome Library Kit was used to construct the NGS library,and then the constructed libraries were quantified and quality tested.5.NGS for genotyping of STR and data analysis:Quantitative and qualitatively completed libraries were sequenced using the Illumina Miseq~TMM NGS sequencer.The sequences of samples were identified depending on the adapter sequence at the end of primers for NGS after raw data were obstained.Alleles of 3 STR loci was typed by using STRait Razor V2.0 software based on the Linux system.Prior to running the software,the allele configuration files of 3 STR loci were input to the software.The allelic configuration files includes:loci name,chromosome,forward 5'and 3'flanking sequence,reversed 5'and 3'flanking sequence,forward and reverse sequence of motif,alleles and sizes of locus.6.Statistical analysis:The frequencies of alleles and haplotypes were obtained by using the direct count method,and forensic application parameters included genetic diversity(GD),the power of discrimination(DP)and excluding probability of paternity(PE)and haplotype diversity(HD).Result1.The CE technique was performed DYF404S1,DYF399S1,and D8S1179 three locus typing on 225 unrelated male individuals in Henan Han.DYF404S1 was a double-copy Y-STR locus,and DYF399S1 was a triple-copy Y-STR locus.Alleles of DYF404S1 locus ranged from 11 to 18 in the Henan Han population.A total of 28 genotypes were observed,the most frequent genotype was 14/15 with a probability of 11.9%.There were 3 abnormal alleles showed as tri-allelic patteners.The genetic diversity(GD)and the power of discrimination(DP)of DYF404S1 were 0.940 8 and 0.938 9,respectively.94 haplotypes for the DYF399S1locus were observed,and the range of alleles was 12~22.The most common haplotype was18/18/18.GD and DP of DYF399S1 were 0.987 9 and 0.986 0,respectively.Allelic range of D8S1179 locus was 9~17.A total of 26 genotypes were found and its heterozygosity and DP were 0.892 8,and 0.849 1,respectively2.Allele sequence analysis of 3 loci was performed by using NGS.Firstly,the methods for STR typing of the D8S1179 locus using NGS was studied by adding adapters at both ends of the primers.The 9948 was selected as a positive control DNA to verify the sensitivity of NGS method,at the same time,100 unrelated male individuals were typed by NGS.Consequently,all samples were successfully typed.STR typing of locus DYF404S1 was performed according to the established conditions and methods for NGS typing for STR genetic markers.Among 225 unrelated male individuals in Henan Han population,220 were successfully typed,with a success rate of 97.8%.And NGS typing results were consistent with the result of CE technology.However,DYF399S1 locus was failed in NGS typing due to the possible cause of its long fragment.3.DYF404S1 locus abnormal allele type,3 out of 225 unrelated male individuals in Henan Han were found to have abnormal types,all of which showed tri-allelic patteners and the probability of abnormal typing was 1.3%.The CE profile of 2 samples showed that the peak height and area of the allele were approximately the same.In one sample,the peak height and area of one peak were approximately equal to the sum of the peak heights and peak areas of the other two peaks.Conclusion1.Analysis of genetic polymorphisms of three STR loci including DYF404S1,DYF399S1 and D8S1179 in Henan Han population would provide the basic data for forensic practice and study of human genetics.2.DYF404S1 and D8S1179 loci were used to construct the NGS library using the method of adding adapters at both ends of the primers.Methods for genotyping of multi-copy Y-STR and autosomal STR loci by NGS were successfully established,which has high sensitivity.3.There were 3 samples with allelic abnormalities of DYF404S1a/b locus which showed as tri-allelic patteners in the Henan Han population.